【作者】 齐岩；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2005， 硕士

【摘要】 干旱、高盐和低温是严重影响作物生长发育及产量的3 种不同形式的环境胁迫因子,是影响我国乃至世界农业生产的重大问题。因此,如何提高作物抗干旱、高盐和低温的能力是一个全世界都亟待解决的问题。随着基因工程技术的迅猛发展,人们已将一些抗逆功能基因导入植物,但植物抗逆能力的提高有限,无法满足生产上的需要。目前,抗逆反应中的调控基因已逐渐成为研究热点,如果能将调控基因导入植物,将有望使转基因植物抗逆性状获得综合改良,而进行调控基因的克隆和功能鉴定,并从中筛选出能显著提高植物抗逆性的基因是培育抗逆农作物新品种(系)的重要前提条件,也是植物抗渗透胁迫基因工程研究的关键。本研究构建了分别由E12 强组成型启动子和rd29A 渗透胁迫诱导型启动子调控的OsMAPK4 和OsCDPK7 基因的植物表达载体,并优化了烟草遗传转化体系;利用农杆菌介导法将DREB2A、OsCDPK7 和OsMAPK4 基因导入烟草,获得了转基因植株。通过对T1代的抗逆性检测,分析各基因的抗渗透胁迫功能,探讨这些调节基因在植物抗逆基因工程方面的应用前景,为利用这些调节基因进行抗逆基因工程研究奠定基础。主要研究结果如下: 1. 植物表达载体构建构建了4 个植物表达载体,分别命名为pBM29A、pBME12、pBC729A 和pBC7E12。其中pBM29A 用于OsMAPK4 基因在双子叶植物中的渗透胁迫诱导型表达,pBME12 用于OsMAPK4 基因在双子叶植物中的组成型表达,pBC729A 用于OsCDPK7 基因在双子叶植物中的渗透胁迫诱导型表达,pBC7E12 用于OsCDPK7 基因在双子叶植物中的组成型表达。2. 遗传转化体系优化(1) 确定了不定芽分化阶段Km 的筛选压力为20 mg/L。(2) 确定了最佳除菌剂为阿莫西林:克拉维酸钾(2:1),其使用浓度为100 mg/L。3. 遗传转化利用农杆菌介导法将分别由E12启动子和rd29A启动子调控的DREB2A基因、OsMAPK4基因和OsCDPK7 基因对烟草“龙江911”进行遗传转化,获得了大量的抗性苗。4. 抗性苗PCR 检测获得了转化pB2A29A 质粒的PCR 阳性植株66 株,转化pB2AE12 质粒的PCR 阳性植株17 株,转化pBM29A 质粒的PCR 阳性植株13 株,转化pBME12 质粒的PCR 阳性植株45 株,转化pBC729A 质粒的PCR 阳性植株11 株,转化pBC7E12 质粒的PCR 阳性植株36 株。5. Southern 印迹杂交检测检测了转OsCDPK7 基因的PCR 阳性植株15 株,9 株呈阳性,其中转pBC729A 质粒的更多还原

【Abstract】 Drought,high salt and low temperature are three common stress conditions that adversely affect plant growth and crop production.And they are the important problems which affect the agriculture yield of our country even the world.Therefore how to enhance the crops resist drought,high salt and low temperature ability is the question which one urgently awaits to solve.With the rapid development of the gene engineering technique,some functional genes have been transferred to plants.But the improving is limited.At present the regulatory genes has been the research emphases.If the regulatory genes can be transferred to plants,we maybe gain the plants which had excellence characters.It is the important precondition condition to cloning and identification the function of the regulatory genes. In this study,we used tobacco to analysis the function of DREB2A、OsCDPK7 and OsMAPK4 genes.We constructed several plant expression vectors.And the tobacco was transformed by Agrobacterium Tumefaciens.Transgene plants were obtained,and the generation of T1 was inspected by resistance identification.The function of the targeted genes was primarily analyzed.The main results was summarized as follows: 1. Construction of plant expression vectors There were four plant expression vectors were constructed,they were pBM29A,pBME12,pBC729A and pBC7E12.The plant expression vector pBM29A contained the OsMAPK4 gene,which was expressed in dicotyledonous plant and induced by osmotic stress conduction.The plant expression vector pBME12 contained the OsMAPK4 gene,which was constitutively expressed in dicotyledonous plant.The plant expression vector pBC729A contained the OsCDPK7 gene,which was expressed in dicotyledonous plant and induced by osmotic stress conduction.The plant expression vector pBC7E12 contained the OsCDPK7 gene,which was constitutively expressed in dicotyledonous plant. 2. Optimization of tobacco genetic transformation system 1 Identified the concentration of Km on stage of adventitious buds differentiation:Km concentration is 20 mg/L.2 The optimization of antibiotics is Amoxicillin and Clavulanate Potassiium(2:1)andthe concentration is 100 mg/L. 3. Transformation mediated by Agrobacterium Tumefaciens DREB2A gene,OsMAPK4 gene and OsCDPK7 gene,which were respectively regulated by promoter E12 and rd29A,were transferred into tobacco “Longjiang 911”. And each combination all obtained the massive resistant seedlings. 4. PCR identification of resistent seedlings There were 66 PCR positive plants be obtained,which were transformed with LBA4404(pB2A29A);there were 17 PCR positive plants be obtained,which were transformed with LBA4404(pB2AE12);there were 13 PCR positive plants be obtained,which were transformed with LBA4404(pBM29A);there were 45 PCR positive plants be obtained,which were transformed with LBA4404(pBME12);there were 11 PCR positive plants be obtained,which were transformed with LBA4404(pBC729A);there were 36 PCR positive plants be obtained,which were transformed with LBA4404(pBC7E12). 5. Southern blot identification of PCR positive plants 15 PCR positive plants that were transformed with OsCDPK7 gene were inspected by Southern bolt,and 9 plants were Southern blot positive plants.There was 1 Southern blot positive plant with pBC729A,and 8 Southern blot positive plants with pBC7E12. 18 PCR positive plants that were transformed with OsMAPK4 gene were inspected by Southern bolt,and 6 plants were Southern blot positive plants.There was 2 Southern blot positive plants with pBM29A,and 4 Southern blot positive plants with pBME12. 6. Analysis of genes function The generation of T1 was inspected by resistance identification.The function of the target genes was primarily analyzed.更多还原